Cosmid Cloning: Cell preparation, DNA packaging, and
Protocol taken from Stratagene's Gigapack packaging
extracts instruction manual
Host bacteria (E. coli LE392)preparation:
- Pick one colony from fresh overnight culture on NZY agar plate and
inoculate 50 ml NZY broth supplemented with 0.2% maltose and 10 mM
Grow at 37C, shaking, 4-6 h (do not grow past OD600
Pellet bacteria 2000 rpm for 10 min.
Resuspend cells in one-half original volume sterile
10 mM MgSO4.
Dilute cells to OD600 of 0.5 with sterile 10 mM MgSO4.
Plating bacteria should be no older than 48 h.
- Remove extracts from freezer and place on dry ice. Start thawing
sonic extract (yellow tube) first.
Quickly thaw freeze-thaw extract (red tube) until
just beginning to thaw.
Add DNA immediately (1-4 ml containing 0.1-5 mg) to
freeze-thaw extract. Place on ice.
Quickly add 15 ml sonic extract to DNA-freeze-thaw
Stir or pipet to mix, avoiding introduction of air
Incubate at room temperature for 2 h.
Add 500 ml phage dilution (SM) buffer. Store at 4
C. Supernatant is ready to be titered.
Transfection: titering library
- Make 1/10 and 1/50 dilutions of packaged DNA in SM buffer.
Mix 25 ml of undiluted and diluted samples with 25
ml cells as prepared above. Let stand at room temperature for 30 min.
Add 200 ml LB and incubate 1 h at 37 C, mixing gently
every 15 min.
Spin tubes for 1 min and resuspend pellet in 50-100
ml LB, and spread on L-agar plates containing selective antibiotic.
Amplifying cosmid library
- In a series of small culture tubes or microfuge tubes, mix packaged
DNA with equal volume of host cells. Incubate 30 min at room temperature.
Add 4 volumes LB, incubate 1 h at 37 C, with shaking.
Resuspend and plate as above. Volumes to be plated depends on titer
of packaged DNA and size of plates used.
To store library, pour 3 ml LB onto plate and scrape
colonies into the broth with sterile razor blade. Resuspend cells,
pool, add appropriate antibiotic to cell suspension, and freeze aliquots
at -80 C.